Abstract Despite many advances in AIDS research, including the availability of potent anti-retroviral therapy (ART) that effectively controls virus replication in a large proportion of HIV-infected patients, a treatment that can cure the infection remains elusive. To this end, new approaches are required to eradicate the reservoirs of latently infected cells that persist during ART and are the source of virus reactivation when therapy is interrupted. In the R21 phase of this grant application we propose to use the existing, well-established non-human primate model of SIVmac infection of rhesus macaques (RMs) to validate studies of HIV eradication/functional cure by developing an experimental system in which virus replication is fully and persistently suppressed in vivo by a potent ART regimen (Aim #1). We will then use this validated model to investigate directly in vivo and in multiple organs the anatomic and phenotypical nature of the persistent reservoirs of latently infected cells, with specific focus on the relationship between expression of co-inhibitory molecules (i.e., PD-1, CTLA-4, TIM-3, and LAG-3) and size of the persistent reservoirs (Aim #2). The results of the studies proposed in the R21 part of this application will pave the way for further experiments, to be conducted in the R33 phase of this proposal, in which we will test, in ART-treated SIV-infected RMs with full suppression of virus replication, immune-based interventions aimed at reducing and possibly eliminating in vivo the persisting reservoirs of latently infected cells. The key proposed intervention consists of a blockade of the co-inhibitory pathway most closely associated with SIV latency, which will be performed as a stand-alone therapy or in combination with a non-specific virus reactivating agent (i.e., the histone deacytelase inhibitor, SAHA). We believe that the proposed studies will provide unprecedented insights into the biology of persistent virus reservoirs of latently infected cells, and elucidate the potential of targeting co-inhibitory pathways to reduce the reservoir during SIV infection.